The intracellular messenger, cyclic GMP (cGMP) has diverse physiological roles in a variety of tissues and there is a correspondingly diverse array of human diseases and disorders resulting from misregulation of cGMP signaling pathways. There are also many different proteins that are responsible for the synthesis, degradation and action of cGMP. Although the properties of most of the major components of the cGMP signaling cascade are well known, the precise mechanisms for the temporal and spatial regulation of cGMP levels and action of cGMP within a cell are less well understood. Several members of the cGMP cascade are important targets for the development of therapeutic agents targeting cardiovascular disease and neurological and urogenital disorders. A better understanding of the cyclic GMP signaling dynamics within a cell and the identification of additional regulatory proteins will likely provide additional targets. The long-term goal of this project is to use the powerful genetic tools available in the fruit fly, Drosophila melanogaster, to examine cGMP signaling in a defined set of sensory neurons that act as neuronal oxygen sensors initiating behavioral responses to hypoxia. During previous funding periods of this project, we have shown that the primary molecular oxygen sensors within these cells are the atypical soluble guanylyl cyclases (sGCs) that catalyze the synthesis of cGMP and are directly regulated by oxygen. One of the three aims in this proposal will test the specific steps in the model that we have developed to explain how hypoxia triggers a behavioral escape response via increases of cGMP within a population of sensory neurons. We will test whether optogenetic control of cGMP levels within these cells is sufficient to trigger the behavior. We have also developed a mathematical model for this process and will test this model by measuring the activation of these neurons and increases of cGMP in real time as the animals are subjected to hypoxic stimuli. A final component of the model will be to identify the phosphodiesterase responsible for bringing the cGMP levels back to baseline within the cells. The other aims of this project are to investigate how additional genes interact with this cascade. One of these genes is nitric oxide synthase that generates the gaseous messenger, nitric oxide. Both nitric oxide and oxygen bind to and inhibit the activity of the atypical sGCs, yet appear to have opposite effects on the escape behavior - reduced oxygen activates the behavior, whereas loss of nitric oxide synthase diminishes the behavior. The other genes, named kul and CG10011, have no known function in cGMP regulation. Kul is related to genes that are known to function in non-amyloidogenic metabolism of amyloid precursor protein and CG10011 is related to a gene that causes inherited deaf-blindness. We will use genetic, imaging and behavioral approaches to determine where in the hypoxia/cGMP cascade these genes exert their effects. These studies will yield new insights into the functioning of these genes and a more detailed understanding of cGMP signaling and hypoxia detection. PUBLIC HEALTH RELEVANCE: The intracellular messenger, 3',5' cyclic guanosine monophosphate (cGMP) regulates a wide variety of physiological processes and dysfunction of cGMP regulation leads to a wide variety of human diseases and disorders. The long-term goal of the project described in this proposal is to identify additional regulatory components of cGMP metabolism in identified neurons using the powerful genetic tools available in the fruit fly, Drosophila melanogaster. There are many precedents showing that cellular processes identified in Drosophila have direct counterparts in humans and hence will lead to novel therapeutic targets in the future.